Immuno histo chemistry : Immuno histo chemistry Prepared by:
Dr.Sourab Kumar contents : contents Definition
Basic concepts of immunohistochemistry
Troubleshooting in immunohistochemistry
Marker Proteins used in immunohistochemistry Definitions : Definitions Immunohistochemistry is a technique for identifying tissue constituents (antigens) by means of antigen-antibody interactions.
The site of antibody binding being identified either by direct labeling of the antibody, or by use of secondary antibody labeling method. A.H.Coons : A.H.Coons In 1940, (Microbiologist) described identification of tissue antigens in pneumococci by using direct fluorescence Applications : Applications Cancer diagnostics
Treatment of cancer
Research Alteration of tissue architecture : Alteration of tissue architecture Slide 7: IMMUNOHISTOCHEMISTRY STEPS Tissue sections : Tissue sections TISSUE SECTIONS : TISSUE SECTIONS Fresh tissue biopsy sections
Old Biopsy sections(Registries, Old paraffin blocks)
Fresh biopsies with frozen techniques
Adhesives Slide 11: After tissue sectioning let us start with IHC procedure, that is antigen retrieval. Slide 12: But before that, we have to carry out two steps prior to undertaking the antigen retrieval procedure Blocking Endogenous Enzymes : Blocking Endogenous Enzymes Why??? Blocking endogenous enzymes : Blocking endogenous enzymes 10min Biocare products available for blocking endogenous enzymes : Biocare products available for blocking endogenous enzymes Buffer solutions : Buffer solutions Wash the slide thoroughly with TRIS neutralized buffered solutions 30 Sec Slide 17: Buffer Solutions : Buffer Solutions Slide 19: Now shall we proceed with antigen retrieval procedure???
? Slide 20: NO Blocking background staining : Blocking background staining Why??? Blocking background staining : Blocking background staining Specific. 15min
Immunoglobulins and fibrinogens
Previous infections of animal
Manufacturer Slide 23: Background Staining Buffer solutions : Buffer solutions Wash the slide thoroughly with TRIS neutralized buffered solutions 30 Sec Slide 25: What is antigen Retrieval(Bring back the antigens)?
Where have they gone? Antigen Retrieval Purpose : Antigen Retrieval Purpose Epitopes or antigen binding sites are not clear.
Breaks the formalin induced cross-linking bonds between epitopes and unrelated proteins, thereby allowing better penetration of antibody and accessibility of epitopes. Advantages of antigen retrieval : Advantages of antigen retrieval Enhanced exposure of antigenic epitopes on surface of tissue section.
Allows for reduction in incubation time of primary antibodies or increase dilution of primary antibodies.
Consistent and reliable staining.
Reduces false negatives in formalin-fixed paraffin embedded tissue sections.
Ease of use. Procedure of antigen retrieval : Procedure of antigen retrieval The retrieval techniques of unmasked antigens :
(1) proteolytic enzyme digestion
(3) microwave and trypsin
(4) pressure cooker Proteolytic enzyme digestion : Proteolytic enzyme digestion pH 7.8; 0.1MNaOH; 37degree C Slide 31: Multiple chamber couplin Jar 750W for 10min Slide 32: Microwave Oven 10 min Slide 34: Pressure Cooker 3 Liters ; pH 6.0; 15psi 2min Distributors in india : Distributors in india Allied Scientific Products39/19B Gopal Nagar RoadKolkata-700027India
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Reference: G:\Immunohistochemistry\kolkata distributor.htm Slide 36: Steamer 40min Antibody treatment : Antibody treatment Direct
indirect Direct Method : Direct Method Tissue Antigen Labeled Antibody 30min Two-Step Indirect Method(Most widely used) : Two-Step Indirect Method(Most widely used) Tissue Antigen Primary Antibody Secondary Antibody Types of indirect technique : Types of indirect technique ABC method
PAP and APAAP method
Dextran polymer conjugated two-step visualization system
Immunogold silver staining technique
Hapten labelling technique
Mirror image complementary antibody labelling technique ABC Method (avidin-biotin complex method ) : ABC Method (avidin-biotin complex method ) Slide 55: Enzyme and chromogen substrate added PAP Method (peroxidase anti-peroxidase method) : PAP Method (peroxidase anti-peroxidase method) Slide 57: IGSS technique SP Method(streptavidin peroxidase conjugated method) : SP Method(streptavidin peroxidase conjugated method) Labels : Labels Slide 60: DAB Solutions Chromogens : Chromogens Antigen+Antibody 2nd Antibody+Chromogen(HRP)
Why we have to add chromogens???
What is the necessity??? IHC automatic stainer : IHC automatic stainer Slide 71: IMMUNOHISTOCHEMISTRY STEPS Troubleshooting : Troubleshooting 1. Weak or No Staining
3. High Background High Background : High Background Over-staining : Over-staining Weak or No Staining : Weak or No Staining Weak or No Staining : Weak or No Staining ANTIBODIES APPLIED TO THE STUDY OF SMALL ROUND-CELL TUMORS : ANTIBODIES APPLIED TO THE STUDY OF SMALL ROUND-CELL TUMORS KERATIN S100
MYOGENIN ANTIBODIES APPLIED TO THE STUDY OF LARGE POLYGONAL-CELL TUMORS : ANTIBODIES APPLIED TO THE STUDY OF LARGE POLYGONAL-CELL TUMORS KERATIN S100
CD117 REAGENTS APPLIED TO THE STUDY OF SPINDLE-CELL TUMORS : REAGENTS APPLIED TO THE STUDY OF SPINDLE-CELL TUMORS KERATIN S100 PROTEIN
THROMBOMODULIN CD99 REAGENTS APPLIED TO THE STUDY OF PLEOMORPHIC TUMORS : REAGENTS APPLIED TO THE STUDY OF PLEOMORPHIC TUMORS KERATIN HMB45
S100 PROTEIN CD99 REAGENTS USED IN PARAFFIN SECTION PHENOTYPING OF HEMATOPOIETIC TUMORS : REAGENTS USED IN PARAFFIN SECTION PHENOTYPING OF HEMATOPOIETIC TUMORS CD15 CD79A
CD68 CD163 Slide 82: Epithelium: Keratins
and antibodies to keratins of different molecular weights
Supporting connective tissues:
--Vimentin--fibroblasts, blood vessels
--vWF, CD31 (PECAM)-- endothelial cells of blood vessels
Hematopoeitic tissues: CD45, B220, CD3, F480, Mac-1, Gr-1, CD41
Muscle: desmin, smooth muscle actin
Neural: GFAP, NeuN, F480/Mac-1, MBP, NSE, S100
Hormones: specific antibodies--insulin, casein, etc.
Germ cells: alpha-feto protein (teratomas)
Proliferation markers-Ki-67 Cytokeratins : Cytokeratins Epithelial marker protein HMB 45 : HMB 45 S-100 : S-100 . P 63 : P 63 Ki 67 : Ki 67 High-grade fibrosarcoma. Spindle-shaped lesional cells have blunt-ended nuclei and here are surrounded by minimal fibrous stroma. Many mitotic figures are seen(p53) : High-grade fibrosarcoma. Spindle-shaped lesional cells have blunt-ended nuclei and here are surrounded by minimal fibrous stroma. Many mitotic figures are seen(p53) HPV : HPV References : References Bancroft histological procedures
Culling histological procedures4. Journals of Oral Pathology and Medicine
5. Shafer’s oral pathology Thank you ! : Thank you !